Figure 2.

scd Mutants Display Post-Golgi Trafficking Defects.

Quantitative time-lapse analysis of the number and intensity of PIN2-GFP-labeled BFA bodies in wild-type, scd1, and scd2 seedling root cells pretreated with BFA ([A] to [O]) and BFA plus CHX ([P] to [R]) followed by removal of the inhibitors.

(A) to (L) Trafficking of intracellular PIN2-GFP following BFA washout in wild-type ([A] to [D]), scd1-2 ([E] to [H]), and scd2-1 ([I] to [L]) root cells. Seedlings were treated with 50 µM BFA for 60 min prior to washout with 0.5× MS and imaged at 0, 20, 40, and 60 min by CLSM. Arrowheads indicate PIN2-GFP-labeled BFA bodies. Bar = 10 µm.

(M) The number of PIN2-GFP-labeled BFA bodies per cell after BFA removal (minimum of 872 cells counted from a minimum of 27 roots each). Shown are means ± sd. **P < 0.01 and ***P < 0.001 (t test, compared with wild-type value).

(N) PIN2-GFP BFA body intensity/area. Total PIN2-GFP BFA body intensity was determined and divided by the BFA body area per cell (minimum of 163 cells from a minimum of 9 roots). Arbitrary intensity units. Shown are means ± sd. **P < 0.01 and ****P < 0.0001 (t test, compared with wild-type value). Seedlings were treated as described in (A) to (L).

(O) Number of BFA bodies prior to washout in Col-0 and scd mutants without CHX. Shown are means ± sd.

(P) Number of BFA bodies prior to washout in Col-0 and scd mutants in the presence of CHX. Shown are means ± sd. ****P < 0.0001 (t test, compared with wild-type value)

(Q) Relative number of PIN2-GFP-labeled BFA bodies per cell in the presence of CHX after BFA removal (minimum of 404 cells counted from a minimum of 13 roots each). Shown are means ± sd. *P < 0.05, ***P < 0.001, and ****P < 0.0001 (t test, compared with wild-type value). Seedlings were pretreated with 50 μM CHX for 30 min and then treated with 50 µM BFA and 50 μM CHX for 60 min prior to washout with 0.5× MS and imaged at 0, 20, 40, and 60 min using CLSM. Total number of BFA bodies per cell was normalized with the number of BFA bodies per cell at the start of BFA washout for each genotype.

(R) PIN2-GFP BFA body intensity/area in the presence of CHX. Total PIN2-GFP BFA body intensity was determined and divided by the total BFA body area per cell (minimum of 100 cells from a minimum of 9 roots each). Arbitrary intensity units. Shown are means ± sd. ****P < 0.0001 (t test, compared with wild-type value). Seedlings were treated as described in (Q).

(S) to (U) PIN2-GFP in Col-0, scd1-2, and scd2-1 in untreated root cells. Bar = 10 µm.